苏淮猪背最长肌FAPs细胞体外成脂能力及其基因表达模式的研究

doi:10.11843/j.issn.0366-6964.2023.10.012

摘要/Abstract

摘要： 旨在体外获得纯度较高的苏淮猪背最长肌纤维/脂肪形成祖细胞（fibro/adipogenic progenitors，FAPs），并评价其在体外传代过程中成脂能力与基因表达模式的变化。本研究取1日龄苏淮公猪背最长肌，消化获得悬浮混合细胞，利用抗表面特异抗原血小板衍生生长因子受体α（PDGFRα）抗体通过免疫荧光评价4种不同差速贴壁时间分离的FAPs细胞的纯度，确定最佳差速贴壁时间。然后比较不同代数FAPs细胞（P1、P3和P5代）的体外成脂分化能力，并利用RNA-Seq比较不同代数FAPs细胞的基因表达模式，鉴别差异表达基因、KEGG通路与GO功能分类。研究结果表明，差速贴壁30 min分离的苏淮猪背最长肌FAPs细胞的纯度较高。对不同代数FAPs成脂能力评价发现，随着体外传代次数的增加，其成脂能力显著下降。利用RNA-Seq发现P3和P5代FAPs细胞的基因表达模式相近，P3和P5代分别与P1代FAPs细胞相比，鉴定到2 336个共有的差异表达基因，其中差异上调基因1 102个，差异下调基因1 234个，KEGG和GO分析表明共有的差异上调基因主要富集在PI3K-AKT-FoxO、PI3K-AKT-HIF-1和cGMP-PKG等通路，共有的差异下调基因主要富集在DNA复制和修复等通路，其中可抑制细胞增殖与成脂分化的IRS1、FOXO3、CCNG2、EGFR、FGF1基因表达上调，可促进细胞增殖的CSF1R和SIPA1L2基因表达下调。P3与P5相比发现，差异上调基因主要富集在NOD-like、MAPK和非典型Wnt信号等通路；差异下调基因主要富集在VEGF、PPAR、Notch以及IL-17等信号通路，其中可抑制细胞成脂分化的TNFAIP3、AREG、FGF1、FGF9基因表达上调，可促进成脂分化的PPARγ、C/EBPα、LCN2基因下调。本研究结果表明，差速贴壁30 min能够得到纯度较高的猪FAPs细胞，FAPs细胞在体外培养过程中成脂分化能力不断下降，其基因表达模式发生了较大变化，主要表现为可促进细胞增殖和成脂分化的信号通路及其相关基因表达水平下降，本研究结果可为开发能维持猪FAPs细胞体外成脂能力的培养基配方提供参考。

关键词: 猪, FAPs细胞, 体外, 成脂能力, 基因表达

Abstract: This study aimed to obtain higher purity of fibro/adipogenic progenitors (FAPs) from longissimus dorsi muscle of Suhuai pig, and evaluate the alteration of the adipogenic capacity and gene expression patterns during in vitro culture. The longissimus dorsi muscle of one-day Suhuai boar was digested to obtain suspended mixed cells. In order to determine the optimal differential adhesion time for FAPs cell, the purity of cell isolated from 4 different adhesion time were evaluated by immunofluorescence using an antibody against platelet-derived growth factor receptor α (PDGFRα). Then, the adipogenic differentiation ability of different passage of FAPs cells (P1, P3 and P5) cultured in vitro was compared, and the RNA-Seq was used to identify the differentially expressed genes (DEGs), KEGG pathways and GO functional classification in FAPs cells with different passages.The results showed that the purity of FAPs cells isolated from longissimus dorsi muscle of Suhuai pig by differential adherence for 30 min was much higher than other conditions. Evaluation of the adipogenic capacity of FAPs cells with different passages showed that the adipogenic ability of FAPs cells decreased significantly with the increase of passages in vitro. RNA-Seq results showed that the gene expression patterns of P3 and P5 generation FAPs cells were similar. Compared with P1 to P3 or P1 to P5 generation cells respectively, a total of 2 336 common DEGs was identified, including 1 102 up-regulated DEGs and 1 234 down-regulated DEGs. KEGG and GO analysis showed that the common up-regulated DEGs were mainly enriched in PI3K-AKT-FoxO, PI3K-AKT-HIF-1 and cGMP-PKG signaling pathways etc., and the common down-regulated DEGs were mainly enriched in DNA replication and repair signaling pathways etc. Among them, the genes of IRS1, FOXO3, CCNG2, EGFR and FGF1, which could inhibit cell proliferation and adipogenic differentiation were up-regulated, while the genes of CSF1R and SIPA1L2, which could promote cell proliferation were down-regulated. Compared P3 with P5 generation cells, the up-regulated DEGs were mainly enriched in NOD-like, MAPK and non-canonical Wnt signaling pathways etc.; The down-regulated DEGs were mainly enriched in VEGF, PPAR, Notch and IL-17 signaling pathways etc. Among them, TNFAIP3, AREG, FGF1 and FGF9 genes, which could inhibit adipogenic differentiation were up-regulated, and PPARγ, C/EBPα and LCN2 genes, which could promote adipogenic differentiation were down-regulated.These results showed that porcine FAPs cells with higher purity can be obtained by differential adherence for 30 min. The adipogenic capacity of porcine FAPs is continuously decreased during in vitro culture, and the gene expression patterns also undergo significant changes during in vitro culture. The major change for in vitro cultured FAPs is the reduction of gene expression related to cell proliferation and adipogenic differentiation. This study could provide a reference for developing a medium formula, which could maintain the adipogenic capacity of porcine FAPs cells during in vitro culture.

Key words: pig, FAPs cell, in vitro, adipogenic capacity, gene expression

中图分类号:

S828.2

季铮渝, 倪梦茹, 张兆博, 赵赶, 黄赞, 李平华, 黄瑞华, 侯黎明. 苏淮猪背最长肌FAPs细胞体外成脂能力及其基因表达模式的研究[J]. 畜牧兽医学报, 2023, 54(10): 4126-4142.

JI Zhengyu, NI Mengru, ZHANG Zhaobo, ZHAO Gan, HUANG Zan, LI Pinghua, HUANG Ruihua, HOU Liming. Research on Adipogenic Capacity and Gene Expression Pattern of in Vitro FAPs Cells Derived from Longissimus Dorsi Muscle of Suhuai Pig[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4126-4142.

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